This project will study structure/function relationships of mammalian cardiac myosin. Myosin is the protein that, through its interaction with actin, is the force generator of the heart. A detailed knowledge of mammalian cardiac myosin will be important in understanding the function of normal and diseased hearts. The primary goal of this study is to define the domains within the myosin heavy chain that underlie the functional differences between the alpha and beta cardiac myosin heavy chain isoforms. (This will involve the construction of alpha/beta myosin heavy chain hybrids.) Recently, it has been shown that the myosin molecule itself is defective in patients with hypertrophic cardiomyopathy. Thus in addition to delineation of basic structure/function relationships of mammalian cardiac myosin, the methodology developed in this project will also be used to understand mutations in myosin that lead to this human heart disease. In order to study cardiac myosin, we are developing two expression and functional assay systems. The first entails myosin expression in a cultured cell line derived from insects (SF9 cells) that can be infected with a virus (baculovirus) altered in the laboratory so that it will produce large amounts of mammalian cardiac myosin. This myosin will be isolated and its interactions with actin will be studied. The second approach involves the transfer of DNA that codes for cardiac myosin into cultured avian myogenic cells. The cells will remain in culture until they produce and incorporate the mammalian cardiac myosin. A single cell will then be removed from the culture dish, and its structural and contractile properties will be examined. In this manner it will be possible to document how changes in the myosin molecular alter both the structural and contractile properties of cultured muscle cells and thus the cells of the human heart.